Mitochondrial Sirtuin-3 (Sirt-3) is a member of the class III histone deacetylases, which possess nicotinamide adenine dinucleotide (NAD+) dependent deactylase and adenosine diphosphate (ADP) ribosyltransferase activities. In the last decade, Sirt-3 has become a highly promising target for curing mitochondrial disorders. This current study examined the effect of long term habitual exercise on the expression of Sirt-3 in plantaris muscle and its correlation with mitochondrial content indicated by the level of Cytochrome c oxidase subunit-4 (Cox-4) protein. 20-week habitual exercise significantly increased the Sirt-3 expression (P < 0.05) and positively correlated with Cox-4 expression in plantaris muscle (P < 0.05). It indicated that Sirt-3 contributed to mitochondrial biogenesis after long term voluntary exercise. It was surprising to demonstrate that Sirt-3 was significantly correlated with the transformation of muscle fibre form type-IIb/IIx to IIa (P < 0.05). Collectively, prolonged habitual exercise induced Sirt-3 could possibly promote both mitochondrial biogenesis and fibre type transformation.